ApplicationNo. 615044 filed on 09/19/1975
US Classes:435/135, Carboxylic acid ester435/261, Separation of micro-organism from culture media435/911, Using fungi435/913, Aspergillus435/923, Candida lipolytica435/933Penicillium
ExaminersPrimary: Tanenholtz, Alvin E.
Attorney, Agent or Firm
DescriptionThe present invention relates to a method for microbiologically reducing zearalenone and related compounds. More particularly, the present invention relates to a method for microbiologically reducing the ketonegroup of zearalenone and related compounds (hereinafter sometimes called keto-compounds) to a hydroxyl group. The nomenclature used herein conforms to that described in an article in Tetrahedron Letters, Pergamon Press, Ltd., No. 27, pp. 3109-3114(1966).
The zearalenone compounds which may be reduced by the method of this invention are represented by the formula ##STR1## wherein B is a six-carbon ring which may be saturated, unsaturated, or aromatic and may be substituted with one or more membersselected from the group consisting of hydrogen; hydroxy; lower alkoxy of from 1 to about 6 carbon atoms, such as methoxy, ethoxy, propoxy, pentoxy and the like; lower alkanoyloxy of from 1 to about 6 carbon atoms, such as formyloxy, acetoxy, butyroyloxy,and the like; monocyclic aryloxy of about 6 to 8 carbon atoms, such as phenyloxy, tolyloxy, etc.; and monocyclic aralkyloxy, that is, an alkoxy group having an aryl substituent thereon, wherein the alkoxy portion has 1 to about 5 carbon atoms and thearyl portion has about 6 to 8 carbon atoms such as benzyloxy, tolyl methoxy and the like; and Y is selected from the group consisting of --CH2 --CH2 -- and --CH=CH--. The compounds are reduced to hydroxy-compounds of the formula ##STR2##wherein the substituents have the same meanings as described above.
This invention is particularly advantageous as a method for reducing keto-compounds of the formula ##STR3## to hydroxy-compounds of the formula ##STR4## wherein R and R1 may be the same or different and each is selected from the groupconsisting of hydrogen, hydroxy, and --OR2, wherein R2 is selected from the group consisting of lower alkyl of from 1 to about 6 carbon atoms, monocyclic aryl of about 6 to 8 carbon atoms, and monocyclic aralkyl wherein the alkyl portion has 1to about 5 carbon atoms and the aryl portion has about 6 to 8 carbon atoms; and Y is selected from the group consisting of --CH2 --CH2 -- and --CH=CH--.
Exemplary of the method of this invention are the reductions of zearalenone to zearalenol (wherein B is an aromatic ring substituted in the 2 and 4 positions with hydroxyls, and Y is --CH=CH--), zearalanone to zearalanol (wherein B is an aromaticring substituted in the 2 and 4 positions with hydroxyls, and Y is --CH2 --CH2 -- ), dideoxyzearalanone to dideoxyzearalanol (wherein B is an unsubstituted aromatic ring, and y is --CH2 --CH2 -- ), dimethyl zealenone todimethylzearalenol (wherein B is an aromatic ring substituted in the 2 and 4 positions with methoxy groups, and Y is --CH=CH--), tetrahydrozearalanone to tetrahydrozearalanol (wherein B is a saturated ring substituted in the 2 and 4 positions withhydroxyls, and Y is --CH2 --CH2 -- ), etc.
The keto-compounds which may be reduced by the method of this invention may be prepared from zearalenone, which is represented by the following structural formula: ##STR5## Zearalenone is a natural fermentation product which may be produced bycultivating a zearalenone-producing strain of the microorganism Giberella zeae on a suitable nutrient medium as described by Andrews, F. N., et al, U.S. Pat. No. 3,196,019, July 20, 1965, incorporated herein by reference. Preparations ofketo-compounds in which B is a substituted aromatic ring are disclosed by Hodge, E. B., et al., U.S. Pat. No. 3,239,341, Mar. 8, 1966, incorporated herein by reference. Preparations of compounds in which B is an unsubstituted aromatic ring aredisclosed by Wehrmeister, H. L., et al, U.S. pat. application Ser. No. 302,414, filed Oct. 31, 1972, now U.S. Pat. No. 3,887,583 which is incorporated herein by reference. Keto-compounds in which B is a saturated ring are described by Abbott, R.L., U.S. Pat. No. 3,373,037, which issued Mar. 12, 1968, incorporated herein by reference.
The hydroxy-compounds which are the products of the method of this invention exhibit estrogenic activity or aid in increasing the rate of growth of meat-producing animals, e.g. cattle, lamb, and swine. The compounds can be administered toanimals by any suitable method including oral and parenteral administrations. For example, the compound can be blended with ordinary feed containing nutritional values in amounts sufficient to produce the desired rate of growth and can thus be feddirectly to the animals, or the compound can be suspended in a suitable injection suspension medium such as peanut oil and injected parenterally. The amount of compound administered to an animal varies depending upon the animal, desired rate of growth,and the like.
The reduction of the ketone group of zearalenone and related compounds has heretofore been accomplished by chemical means. The reduction has been accomplished, for example, by catalytic reduction with hydrogen in the presence of Raney nickel. Hodge, E. B., et al., U.S. Pat. No. 3,239,345, Mar. 8, 1966, describes such a reduction. When the ketone group of naturally produced zearalenone, or a derivative thereof, is chemically reduced, the resulting hydroxy-compound has two asymmetric carbonatoms, one of which (at the 10' position) exists in only one isomeric configuration, and the other (at the 6' position) exists in either of two isomeric configurations. Thus, the hydroxy-compound exists in two diastereoisomeric forms. The twodiastereoisomeric forms differ somewhat in physical properties, such as melting points, and in biological activity. The diastereoisomer having the higher melting point has generally been preferred, particularly in compounds used to increase the rate ofgrowth in meat producing animals. Various reductive methods have been devised to enhance the concentration of this diastereoisomer in the reduction product, however, no known chemical reductive method yields only the desired form. Accordingly, there isa need for a method for stereospecifically reducing zearalenone and related compounds to hydroxy-compounds in which the hydroxyl group in the 6' position has the desired configuration.
In accordance with the invention, there has been discovered a method for reducing a keto-compound of the formula ##STR6## to a hydroxy-compound of the formula ##STR7## wherein B is a six-carbon ring which may be saturated, unsaturated, oraromatic and may be substituted with one or more members selected from the group consisting of hydrogen, hydroxy, lower alkoxy of from 1 to about 6 carbon atoms, lower alkanoyloxy of from 1 to about 6 carbon atoms, monocyclic aryloxy of about 6 to 8carbon atoms, and monocyclic aralkyloxy wherein the alkoxy portion has from 1 to about 5 carbon atoms and the aryl portion has about 6 to 8 carbon atoms and Y is selected from the group consisting of --CH2 --CH2 -- and --CH=CH--, whichcomprises cultivating a ketone-reducing, enzyme-producing microorganism in a nutrient fermentation medium and contacting the keto-compound with the ketone-reducing enzyme produced by the microorganism under ketone-reducing conditions to reduce theketo-compound to produce the hydroxy-compound.
The method of the present invention comprises subjecting a keto-compound to the enzyme action of a viable culture of a ketone-reducing, enzyme-producing microorganism. Any microorganism which produces an enzyme which reduces the ketone group ofzearalenone and related keto-compounds to a hydroxyl group may be employed. Such microorganisms may be obtained from depositories such as the Northern Regional Research Laboratories, Peoria, Illinois or the American Type Culture Collection, Washington,D.C., or the microorganisms may be isolated from natural sources such as soil samples. Methods for isolating and storing such microorganisms, are generally known by skilled microbiologists. Specific microorganisms which have been found effective inreducing keto-compounds are Penicillium frequentans, ATCC 11080; Aspergillus quadrilineatus, ATCC 12067; Penicillium rubrum, ATCC 10520; Syncephalastrum elegans, ATCC 20471; Chaetomium globosum, ATCC 20470; Candida lipolytica, ATCC 8662; and Rhodotorularubra, ATCC 4558. The ATCC numbers refer to the catalog numbers assigned to the microorganisms on deposit at the American Type Culture Collection. The preferred microorganism for the method of this invention is a yeast, Rhodotorula rubra.
The microorganism which is chosen for the reduction is advantageously grown and stored on a suitable agar slant culture. The composition of the medium used for the slant culture is not critical so long as it is sterile and contains adequatenutrients and growth factors to support the microorganism. A suitable slant medium has been found to be Bennett's Agar, which contains the following ingredients:
______________________________________ Beef Extract 1 g NZ Amine A* 2 g Yeast Extract 1 g Glucose 10 g Agar 20 g H2 O to one liter ______________________________________ *pancreatic digest of casein
The reduction is carried out by growing the microorganism in a nutrient fermentation medium and exposing the keto-compound to the reductive enzyme action of the microorganism.
Suitable nutrient fermentation media are aqueous mixtures which contain assimilable sources of nitrogen and carbon, and growth-promoting amounts of growth factors such as minerals and vitamins. Examples of assimilable nitrogen sources areproteins, peptonized beef broth, urea, asparagine, ammonium salts such as ammonium nitrate and ammonium fumarate, glutamine, glycine, and pancreatic digest of casein. The preferred nitrogen source, either as the sole source, or used in conjunction withothers, is a pancreatic digest of casein such as NZ Amine Type A purchased from Sheffield Chemical Division of National Dairy Products, Norwich, New York. The nitrogen source is present in the medium in growth-promoting amounts. Satisfactoryconcentrations of a pancreatic digest of casein, for example, range from about 0.5 grams per liter to about 100 grams per liter, preferably about 10 to 30 grams per liter of fermentation medium.
The assimilable carbon source is advantageously glucose, e.g. reagent grade glucose or Cerelose, a white, crystallized, refined glucose. Other suitable, but less preferred carbon sources are other carbohydrates which will not deleteriouslyeffect the reduction of keto-compounds, such as xylose, fructose, sucrose, maltose, glycerol, sorbitol, and galactose. As an alternative to supplying actual glucose to the fermentation medium, a precursor thereof, such as starch, which under theconditions of the method, will be converted to glucose, can also be used. The assimilable carbon is added to the fermentation medium in growth-promoting amounts. Glucose is generally added to the medium at a concentration of from about 1 gram to about200 grams per liter or more of fermentation medium, preferably from about 50 grams to about 60 grams per liter of fermentation medium.
Minerals such as magnesium, sodium, potassium, and iron salts of phosphates, chlorides, sulfates, etc. and other growth factors, such as vitamins, e.g. B. vitamins; coenzymes; and other organic substances are advantageously added to the medium. Important vitamins and various other growth factors, including certain minerals, are advantageously introduced into the medium via a yeast extract. The yeast extract is usually present in amounts of from about 0.1 gram per liter to about 10 grams perliter, preferably about 1 to 4 grams per liter of fermentation medium. If a pancreatic casein digest is employed as the nitrogen source, certain minerals and vitamins are also provided by it. A particularly preferred fermentation medium contains, inone liter, glucose (Cerelose), 55 grams; pancreatic casein digest (NZ Amine Type A), 20 grams; yeast extract, 2 grams; and distilled water, q.s.
Other non-deleterious ingredients such as antifoaming agents, e.g. silicone foam inhibitors, corn oil, lard oil, mineral oil, etc. may be added to the fermentation medium in the desired amounts.
A suitable size aliquot of the fermentation medium is transferred to a container such as an Erlenmeyer flask, which is then capped with a filter e.g. cotton, to prevent contaminating bacteria from entering and is preferably sterilized prior toinoculation, for instance by autoclaving or subjecting to steam at superatmospheric pressures. After sterilization, the medium is inoculated by transferring to it an inoculum containing a viable culture of the microorganism from, for instance, an agarslant or other source.
The keto-compound may be introduced into the fermentation medium prior to inoculation, or at any time during the growth of the microorganism, however, the preferred technique is to add the keto-compound after substantial growth has occurred. Themedium is incubated at a temperature sufficient for optimal growth, for a time sufficient to provide a substantial cell concentration for the reduction. Incubation temperatures are generally from about 10° C to about 60° C, preferablyabout 20° C to 35° C. A substantial cell concentration is preferably achieved prior to the addition of the keto-compound. Such a cell concentration is generally obtained after from about 16 hours to about 30 hours preferably about 22 to26 hours from the time of inoculation.
After a suitable growth has been obtained the keto-compound may be added to the fermentation medium. The keto-compound may be added in any convenient form, but preferably is added from a solution, e.g. an alcohol solution of the compound. Theketo-compound is introduced into the fermentation medium at a concentration sufficiently high to provide optimal reduction efficiency, but not too high to cause significant amounts of unreacted ketocompound to remain in the medium. Concentrations offrom about 0.001 mg/100 ml to about 200 mg/100 ml, preferably about 50-100 mg/100 ml of fermentation medium provide satisfactory results. The lower the concentration of the keto-compound, the more complete is the reduction; however, based on the cost ofthe medium, recovery, etc., optimum overall efficiency occurs at higher concentrations. The solubility of the keto-compound in the aqueous fermentation medium limits, to some extent, the maximum concentration at which the keto-compound can be reducedefficiently. Various means may be employed to increase the solubility of the keto-compound in the medium. Such means include the formation of a more soluble complex or salt of the compound, or controlling the pH of the medium. The preferred method ofincreasing the solubility of the keto-compound is by controlling the pH of the fermentation medium in a range of from about 6 to about 11.5, preferably about 7 to about 10.5. The "controlling" may or may not involve an actual pH adjustment, e.g. anincrease of the pH of the medium. If, however, an upward adjustment of the pH is required, this may be accomplished by the addition of a suitable pH-increasing base such as sodium hydroxide or ethylenediamine to the fermentation medium in amountssufficient to achieve the desired pH.
The keto-compound is exposed to the reducing action of the microorganism for a time sufficient to provide substantial reduction. Generally from about 3 hours to about 36 hours is adequate, about 20 to 28 hours being preferred.
The reductive microorganisms used in this method generally function either aerobically or anaerobically. It has been found, however, that optimum results are obtained when the growth stage, i.e. the period between inoculation and addition of theketo-compound, is conducted aerobically and the reduction stage, i.e. after addition of the keto-compound, is conducted anaerobically. This technique is advantageously accomplished by agitating the fermentation medium during the growth stage, forinstance, by shaking or sparging with air, and sealing the vessel containing the fermentation from air during the reduction stage.
The reduction of the ketone group by the method of this invention occurs through the action of an enzyme or combination of enzymes produced intracellularly by the microorganism. For this reason, the efficiency of the reduction may be improved byincreasing the effective concentration of the enzymes produced by the microorganism. Frequently, the concentration of the enzymes is increased by a factor of 1 to about 3 times prior to the introduction of the keto-compound. This effect may beaccomplished by enhancing the cell concentration, for instance, by centrifuging a portion of the fermentation medium, and resuspending the concentrated cells in another portion of whole medium. This technique provides improved reduction efficiencies,for instance, by doubling the cell concentration, an approximate 20% increase in the percentage of keto-compound reduced may result. Another method of effectively increasing enzyme concentration is by disrupting the cells to free the enzymes, thusproviding greater exposure of the keto-compound to the action of the enzymes. In this case, the disrupted cells may be removed and the cell-free enzyme solution may be used for the reduction.
After the reduction has been accomplished, the product may be recovered by any suitable method. A suitable recovery method comprises acidifying the fermentation medium to a pH of from about 1.5 to about 5 with a mineral acid, e.g. HCl or H2SO4 ; extracting the acidified fermentation medium with a suitable water-immiscible solvent in which the keto-compound is soluble such as chloroform or methylene chloride; and removing the water-immiscible solvent to leave a residue containing thereduction product. The product may be further purified, for instance, by recrystallization or distillation depending upon the particular product formed.
It is apparent, therefore, that a method has been discovered for stereospecifically reducingzearalenone and related keto-compounds to hydroxy-compounds by the action of a ketone-reducing microorganism. The invention is further illustrated by following examples which are not intended to limit the invention.
All of the microorganisms described in the following examples were stored on Bennett's Agar slant cultures.
Several cultures from various sources were tested by the following procedure for their ability to reduce zearalenone. A fermentation medium having the following composition was prepared:
______________________________________ Cerelose 55 g NZ Amine A 20 g Yeast Extract 2 g Distilled Water to 1 liter ______________________________________
The medium was dispensed in 100 ml quantities into 500 ml Erlenmeyer flasks. The flasks were capped with two milk filters and one sheet of Kraft paper (paper removed before inoculation). The flasks of medium were autoclaved at fifteen poundssteam pressure for fifteen minutes.
Flasks of the medium were routinely inoculated from slant cultures and incubated at 30° C on a rotary shaker. After suitable growth in the fermentation medium (usually 24 hours), one ml of an alcoholic solution of zearalenone (50 mg ofzearalenone/ml of ethanol or methanol) was aseptically added. The flasks were incubated at 30° C for 72 hours on a slow shaker.
For detecting ability to reduce zearalenone, the whole cultures were adjusted to pH 2 with concentrated hydrochloric acid and shaken vigorously with 50-100 ml of chloroform. The resulting mixture was filtered through filter paper to remove cellsor mycelium. The filtrate was poured into a separatory funnel and the phases were allowed to separate. The chloroform layer was drawn off into a flask and a portion was evaporated on a steam bath. The residue was dissolved in one ml of chloroform andspotted on silica gel thin-layer chromatography plates. The TLC plates were developed in chloroform:glacial acetic acid (90:10) or cyclohexane:ethyl acetate (2:1). The developed plates were air dried and viewed under short and long wave ultravioletlight. Zearalenone and zearalenol standards were spotted on the plates as markers. Reduction was indicated by a fluorescing and quenching spot with the Rf of zearalenol. Some of the cultures which exhibited the ability to reduce zearalenone wereidentified to be Penicillium frequentans, Aspergillus quadrilineatus, Penicillium rubrum, Syncephalastrum elegans, Chaetomium globosum, Candida lipolytica, and Rhodotorula rubra.
One hundred milliliters of fermentation medium prepared as described in Example I, was transferred to a 500 ml Erlenmeyer flask. The flask was capped with two milk filters and one sheet of Kraft paper (paper removed before inoculation). Theflask was autoclaved at 15 lb. steam pressure for 15 minutes and cooled to about 30° C. The medium was inoculated with a culture of the yeast Rhodotorula rubra and incubated under vigorous agitation for 24 hours at 30° C. An alcoholicsolution of zearalenone (1 ml, 50 mg zearalenone) was aseptically added, and the medium was further incubated for 72 hours without agitation. The medium was then shaken with 50 ml of methylene chloride and centrifuged. The lower, methylene chloridelayer was withdrawn with a syringe and evaporated to dryness. The residue was redissolved in one ml of methylene chloride and spotted on a thin-layer chromatography plate. The thin-layer plate was developed in chloroform:glacial acetic acid (90:10). The zearalenone and zearalenol bands were scraped off the plate and separately eluted with methanol. Since the two compounds have approximately the same U.V. maxima, and extinction coefficients, the % conversion was calculated as follows: ##EQU1## Theexperiment resulted in about a 44% conversion of zearalenone to zearalenol.
The experiment of Example II was repeated in all essential details except varying concentrations of zearalenone were reduced. The following table summarizes the results.
______________________________________ % Conversion Zearalenone Added of zearalenone mg/100 ml of medium to zearalenol ______________________________________ 6.25 78.9 12.5 84.7 25 83.2 75 24.4 100 17.1 ______________________________________
The experiment of Example II was repeated in all essential details, except the pH of the fermentation medium was adjusted to 8.5 with an aqueous solution of ethylenediamine prior to addition of zearalenone. A 70% conversion was achieved.
The experiment of Example II was repeated in all essential details except the pH of the fermentation medium was adjusted to 8.5 with an aqueous solution of sodium hydroxide prior to the addition of zearalenone. A 67% conversion was achieved.
Media containing various cell concentrations were prepared to determine the effect on reduction efficiency. Fermentation media were prepared, inoculated, and incubated for 24 hours at 30° C as described in Example II. Cellconcentrations were reduced from the normal level by centrifuging portions of the cells out. Higher cell concentrations than normal were achieved by resuspending cells which were centrifuged out of media back into whole medium. The reduction stage ofthe fermentation was conducted as described in Example II. The results are summarized in the following table.
______________________________________ Cell Concentration (% of normal concentration of whole medium) % Conversion ______________________________________ 25% 19.2 50% 39.1 75% 47.7 100% 65.3 150% 85.6 200% 88.9 ______________________________________
EXAMPLES VII- XII
A series of experiments is conducted in which keto-compounds of the structure ##STR8## are reduced to hydroxy-compounds, wherein the substituents of the starting material of each particular example are shown in the following table. In eachexample, the procedure in Example II is repeated in all essential details with the exception that a different starting material is used. Each example results in satisfactory reduction of the keto-compound to a corresponding hydroxy-compound, having thefollowing structure ##STR9##
______________________________________ Example R R1 Y ______________________________________ VII --H --H --CH2 --CH2 -- VIII --OH --OH --CH2 --CH2 -- IX --OCH3 --OCH3 --CH=CH-- X --O--φ* --OH --CH2--CH2 -- XI --OCH2 φ* --OCH2 φ* --CH=CH-- XII --OCH2 CH2 CH3 --OCH2 CH2 CH3 --CH=CH-- ______________________________________ *φ indicates phenyl